apoptosis and oncotic necrosis apoptosis vs oncotic necrosis initiation execution apoptosis...
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Apoptosis and Oncotic Necrosis: The final common pathways of liver injury
Hartmut Jaeschke
Department of Pharmacology, Toxicology & Therapeutics
University of Kansas Medical Center Kansas City, KS
Fibrosis: Response to Cell Injury
Rockey & Friedman, 2006
Chronic Cell Injury: Apoptosis and Oncotic Necrosis
• cellular condensation (cell shrinkage)
• membrane blebbing, but no loss of integrity
• aggregation of chromatin at the nuclear membrane
• formation of membrane bound vesicles (apoptotic bodies)
• no disintegration of organelles; organelles remain intact
• swelling of the cell and lysis
• loss of membrane integrity
• flocculation of chromatin
• no vesicle formation, complete lysis
• disintegration (swelling) of cell organelles
Morphology
Apoptosis Oncotic Necrosis
Hepatocellular Apoptosis
H & E
Galactosamine (500 mg/kg; 6 hr)
Hepatocellular Oncotic Necrosis and Inflammation
H & E
Bile Duct Ligation 3 days
Apoptotic Cell Death: Caspases
Proenzyme (zymogen)
prodomain large subunit linker domain small subunit
Peptidase 1 496
DED DED Pro-Caspase 8 Initiator Caspase
Death effector domainDED
Peptidase 1 277 Pro-Caspase 3
Effector Caspase
Cysteine-dependent aspartase domainPeptidase
PeptidaseCARD 1 416 Pro-Caspase 9
Initiator Caspase
Caspase recruitment domainCARD
Activation of Caspases Proenzyme (zymogen)
Active Caspase (tetramer)
prodomain large subunit linker domain small subunit
Activation of Caspases Proenzyme (zymogen)
Active Caspase (tetramer)
prodomain large subunit linker domain small subunit
0 20 40 90 120 Time (min)
p32
p11 Fas-Ab Jo-2
Proenzyme
Active Fragment
0
2 0 0
4 0 0
6 0 0
Activation of Caspases
0 20 40 90 120 Time (min)
p32
p11
(ΔF/min/mg protein)
600
400
200
0
*
*
*
DDDD DD
FAS-L FAS-L
DDDD DD
FA D
D FA
D D
FA D
D FA
D D
Cas 8
FAS-L
DDDD DD
FA D
D FA
D D
FA D
D FA
D D
Cas 10
C as
p1 0
Casp 6
C asp
8Bid
Ba xB ak
C
C
C
C
C
C
C
C C
C
C
C C
C
Casp 7 Casp 3A
pa f1
A pa
f1
C as
p 9
C as
p 9
C
C C
C
CAD
En do
G En
do G
CAD
En do
G
A IF
A IF
D ia
bl o
D iablo
C
B ak B ak
B ax
B ax
Caspase Substrates
CAD
CARD
C
C
CARD
DISC
FAS-L FAS-L
dATP
dATP
Fas-mediated Apoptosis Signaling in Hepatocytes
CAD ICADCAD
Bid
Bcl-2
Ba k
C
C
C C
C
C
C
C
C
C
C
C Casp 3
C
C
C C
EndoG EndoG
EndoG
EndoG
AIF
Diablo
C
Caspase 3 SubstratesAIF
cIAPDiablo
Intrinsic Pathway of Apoptosis
Ca
Ca
Ca
Ca
AIF
Diablo
Diablo C
AIF
C
C
C
C
C
C
C C
CC
C
C
BaxBakBax
Bak
AIF Diablo
Ca Ca
Ca
Ca
Ca Ca
Ca Ca
Ca Ca
PTP
Bcl-XL
Bcl-2 Bax
Bax
Bax
28S
eIF2α
PT P
Cathepsin
C
A pa
f1CARD
Apaf1CA R
D
Apaf1 CA RD
A paf1
CARDCasp 9
Ca sp
9C asp
9
C
C C
Bax BakBax
Bak
CaCa Ca
Ca
Casp 9
C C
m-Calpain
Casp 12
p53Bax
Caspase Targets in Apoptosis Casp10
Casp 6
Casp 8
Casp 7
Casp 3
A pa
f1
A pa
f1
C as
p 9
C as
p 9
CARD C
CARD
CARD Casp 2
MDM2Fodrin
Prese- nelin2 Gelsolin
Actin Lamin A
DNA fragmentation and chromatin condensation
Cytoskeleton Cell shape and membrane
blebbing
CAD
Trans- glutaminase
Topo- isomerase
PARPDNA-PKEndoGICAD
Keratin- 18
Gas2
Cell cycle and other
p21
FAK
hnRNP
β-catenin
NuMASREB1
Calpastatin
EMAP II
Rock-1
DNA Fragmentation
1 2 3 4 5 6 7 8 9 10 11 12 13
MW Controls G/ET
0
200
400
600
800
1000
(% Vmax)
0 0 20 40 90 120
600
400
200
800
1000 *
*
*
Time (min)
DNA LadderAnti-Histone ELISA
CADCAD
DNA Strand Breaks: TUNEL Assay
Terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling
GalactosamineGalactosamine (500 mg/kg; 6 hr)(500 mg/kg; 6 hr)
Characteristic Features of Oncotic Necrosis
• Morphology: cell swelling, cell contents release, karyolysis
Mechanism is dependent on the insult
Oncotic Necrosis: Acetaminophen- induced Hepatotoxicity
Cell Swelling, Karyolysis
Cell Contents Release : Plasma ALT > 3000 U/L)
DNA Strandbreaks during Oncotic Necrosis
TUNEL Assay
CV
4 h APAP 6 h APAP
DNA Strandbreaks: Oncotic Necrosis vs Apoptosis
TUNEL Assay
CV
6 h Gal/ET 6 h APAP
DNA Fragmentation: Apoptosis vs Oncotic Necrosis
1 2 3 4 5 6 7 8 9 10 11 12 13
MW Controls G/ET
DNA LadderAnti-Histone ELISA
APAP
D N
A F
ra gm
en ta
tio n
(% c
on tro
l)
0
500
1000
1500
2000
C G/E G/E ZVAD
AAP AAP ZVAD
AAP GSH
*
* * #
#
1 2 3 4 5 6 7 8 9
0 0.5 1 2 3 4 6 G/E
Acetaminophen (h)
p32
p11
Caspase-3 Processing during Acetaminophen Toxicity
Lawson et al., Toxicol Appl Pharmacol 156: 179-86, 1999 Gujral et al., Toxicol Sci 67: 322-8, 2002
Ba xB ak
C
C
C
C
C
C
C
C C
C
C
C C
C
C
C C
C
EndoG
En do
G En
do G
EndoG
En do
G
EndoG
A IF
A IF
D ia
bl o
D iablo
C
B ak B ak
B ax
B ax
AIF
C
Diablo AIF
EndoG
DNA Fragmentation: Nuclear Translocation of Mitochondrial Intermembrane Proteins
Diablo
C C
Nucleus
Mitochondria
Control
5 mM AAP, 6 h
Endonuclease G
Bajt et al., Toxicol Sci 94: 217-225,2006
Cytochrome c
Bax
Bid tBid
Co ntr
ol AP
AP 2
h
AP AP
4 h
Co ntr
ol AP
AP 2
h AP
AP 4
h
Mitochondria Cytosol
APAP-induced Bax and tBid Translocation to Mitochondria
APAP Hepatotoxicity in Bax Gene Knockout Mice
P la
sm a
A LT
(U /L
)
0
1000
2000
3000
4000
5000
6000
7000
*
WT Bax-/-
Bajt et al., J Pharmacol Exp Therap, in press, 2007
APAP Hepatotoxicity in Bax Gene Knockout Mice
P la
sm a
A LT
(U /L
)
0
1000
2000
3000
4000
5000
6000
7000
*
WT Bax-/-
Bajt et al., J Pharmacol Exp Therap, in press, 2007
WT
Bax-/-
NAPQI APAP GSH ↓
Protein Arylation
P4501.
2.
Bax
Cyt c / Smac AIF Endonuclease G
ONOO-
Nucleus
DNA- Strandbreaks
ATP ↓
Caspase Activation
ATP ↓
PARP Activation
DNA Repair
DNA- Fragmentation
Chromatin Condensation
NAD+ Depletion
ATP ↓ ⇐ MPT
? Mechanism of Mechanism of APAP ToxicityAPAP Toxicity
Toxicol Sci 89: 31-41, 2006
O2 – NO
Bax MPT
Protein Nitration
Apoptosis vs Oncotic Necrosis
Initiation Execution
Apoptosis Apoptosis ATP high